The programmatic goals of our work supported by GM040654 have been to elucidate novel biological activities triggered by the EGFR- and/or ErbB2-Rho GTPase signaling axes critical in the development and maintenance of the transformed phenotype. These efforts have involved a combination of biochemical, cell biological, and structural approaches, and more recently mouse models. Recent work has focused on our discovery of an exciting signaling outcome in cancer cells that results in the activation of a key metabolic enzyme, glutaminase C (GAC), which catalyzes the hydrolysis of glutamine to glutamate plus ammonia. This represents a critical step for elevating glutamine metabolism, which together with changes in glycolysis (the Warburg effect), is now widely recognized to be essential for malignant transformation. Thus, understanding the regulatory mechanisms responsible for triggering these metabolic changes is of great interest to the cancer biology and pharmaceutical communities. Given its important role in glutamine metabolism, GAC is likely to be regulated in a number of biological contexts. In this renewal application, we will focus on how GAC is activated in breast cancer cells and its consequences for satisfying their glutamine addiction and the development of the malignant state. We also will delineate the mechanisms of action of a novel group of allosteric GAC inhibitors that we recently discovered. They were responsible for highlighting the connections between different signaling proteins and glutamine metabolism, and thus potentially offer new therapeutic strategies for blocking cancer progression. These efforts will be pursued through the following lines of investigation: 1) Defining the phosphorylation event(s) that leads to GAC activation in breast cancer cells. We will identify the protein kinase(s) involved and the essential phosphorylation site(s) responsible for triggering GAC activation in breast cancer cell lines as well as in cells isolated from human breast tumor samples. 2) Determining the mechanistic basis for a new class of allosteric inhibitors that block GAC activation. We will establish how a newly identified allosteric inhibitor of GAC called 968, and related compounds, bind to the enzyme and block its activation. In particular, we want to determine whether this class of inhibitors binds preferentialy to the inactive state of GAC and/or blocks the ability of GAC to be phosphorylated and thus prevents a key step in its activation. 3) Determining the role of GAC activation in transformation and tumor formation. We will determine whether the activation of this key metabolic enzyme is sufficient to drive transformation, or if its primary role is to work together with oncogenic signaling proteins to sustain the transformed state, using cell and mouse tumor models. These studies should provide new insights into how important metabolic changes in cancer cells are manifested, as well as identify novel targets that could be of real value by serving as sites of intervention against this disease.